1. Field of the Invention
The invention relates to peptides immobilized on glass beads and having N-terminal markers and method of preparation thereof and method of use thereof in carrying out biochemical assays.
2. Information Disclosure Statement
Albericio et al. (Peptides, Walter de Gruyter & Co., Berlin--N.Y., pp. 167-170, 1986) and Buttner et al. (Proceedings of Tenth American Peptide Symposium, pp. 210-211, 1987) describe peptides linked to controlled-pore glass.
Fodor et al. (Science, vol. 251, pp. 767-773, 15 Feb. 1991), which is entitled "Light-Directed, Spatially Addressable Parallel Chemical Synthesis", describes peptides bonded at the carboxy terminals through "a!mino groups at the ends of linkers" to glass plates and having photoremovable protecting groups or fluorescent labels at the amino terminals. Aminopropyl is specifically described as a linker. Nitrovcratryloxycarbonyl is specifically described as a photoremovable protecting group. Fluorescein isothiocyanate is specifically described as a fluorescent labeling agent. The peptides are described as synthesized by solid phase methods. Two pentapeptides, YGGFL and PGGFL, are described as having been prepared in a checkerboard pattern on a glass plate by stepwise synthesis of the common tetrapeptide followed by selective removal of the photoremovable protecting group using a checkerboard mask followed by addition of Y (tyrosyl) to form YGGFL followed by selective removal of the photoremovable protecting group from the areas of the plate where it was masked in the first removal followed by addition of P (prolyl) to form PGGFL. Selective detection of YGGFL by mouse monoclonal antibody 3E7, which does not bind PGGFL, followed by fluorescein-labeled goat antibody to mouse monoclonal antibody 3E7 followed by scanning of the plate with an epifluorescence microscope whereby the squares occupied by YGGFL showed fluorescence is described. By similar technique fluorescent antibody analysis of a 32.times.32 checkerboard array of 1024 peptides ranging in length from 0 to 10 amino acids and each occupying a 400 .mu.m-square area of the plate is also described.
Lam et al. (Nature, vol. 354, pp. 82-84, 7 Nov. 1991) describes peptides linked to resin beads wherein each bead contains a single peptide. Using all of the natural amino acids except cysteine (19), 19 separate reaction vessels and stepwise synthesis beads containing all of the possible pentapeptides thereof (19.sup.5 =2,467,099) were prepared as a mixture, which is referred to as "the peptide-bead library" and which was allowed to react with solutions of acceptor molecules coupled to an enzyme (alkaline phosphatase) or fluorescein. In tests using a monoclonal antibody against .beta.-endorphin and streptavidin as acceptor molecules only a few beads became intensely stained (6 and 23 respectively) and these could be separated manually from the beads which were not intensely stained. The acceptor molecules were removed from the beads and each bead, which contained 50-200 picomole of peptide, was analyzed for the amino acid sequence of its linked pentapeptide using an automated peptide microsequencer. As an anti-.beta.-endorphin ligand the synthetic pentapeptide YGGFQ (Q represents glutaminyl) having a K.sub.i value of 15.0.+-.1.7 nM (the natural pentapeptide YGGFL has a K.sub.i value of 17.5.+-.3.2 nM) was thus identified. As binders of streptavidin 19 of 23 pentapeptides were identified as having the HPQ sequence of amino acids and the remaining 4 of 23 pentapeptides were identified as having the HPM sequence of amino acids.
The Albericio et al. and Buttner et al. references relate only to solid phase synthesis of peptides and do not describe or suggest peptides immobilized on a solid support and designed as tools for biochemical assay. The method of the Fodor et al. reference is limited to assays that can be carried out on the solid surface of a glass plate. The method of the Lam et al. reference depends on interaction of a fluorescent acceptor molecule with a peptide immobilized on a single glass bead and on finding and removing the resulting single fluorescent glass bead from many others in order to determine the immobilized peptide thereof. Neither the Fodor et al. reference nor the Lam et al. reference relates to cleavage of an immobilized peptide or to use of a supernatant solution for biochemical assay. The presently described and claimed invention relates to a peptide immobilized on a glass bead, which is in practice one of many glass beads each having the same immobilized peptide, for use as a substrate for a protease, cleavage by which results in a fluorescent peptide fragment in the supernatant solution and a peptide fragment on the glass bead, either or both of which can be analyzed to determine the extent and site of cleavage and is thus distinguished from the cited references.